Ultrasmall Fe3O4 nanoparticles self-assembly induced dual-mode T1/T2-weighted magnetic resonance imaging and enhanced tumor synergetic theranostics

Individual theranostic agents with dual-mode MRI responses and therapeutic efficacy have attracted extensive interest due to the real-time monitor and high effective treatment, which endow the providential treatment and avoid the repeated medication with side effects. However, it is difficult to achieve the integrated strategy of MRI and therapeutic drug due to complicated synthesis route, low efficiency and potential biosafety issues. In this study, novel self-assembled ultrasmall Fe3O4 nanoclusters were developed for tumor-targeted dual-mode T1/T2-weighted magnetic resonance imaging (MRI) guided synergetic chemodynamic therapy (CDT) and chemotherapy. The self-assembled ultrasmall Fe3O4 nanoclusters synthesized by facilely modifying ultrasmall Fe3O4 nanoparticles with 2,3-dimercaptosuccinic acid (DMSA) molecule possess long-term stability and mass production ability. The proposed ultrasmall Fe3O4 nanoclusters shows excellent dual-mode T1 and T2 MRI capacities as well as favorable CDT ability due to the appropriate size effect and the abundant Fe ion on the surface of ultrasmall Fe3O4 nanoclusters. After conjugation with the tumor targeting ligand Arg-Gly-Asp (RGD) and chemotherapy drug doxorubicin (Dox), the functionalized Fe3O4 nanoclusters achieve enhanced tumor accumulation and retention effects and synergetic CDT and chemotherapy function, which serve as a powerful integrated theranostic platform for cancer treatment.

For the design of dual-mode T 1 /T 2 MRI contrast agent, the Fe 3 O 4 nanoparticles with tunable MRI ability are the mostly used materials.Ultrasmall Fe 3 O 4 nanoparticles with diameters below 5 nm have been recognized to possess T 1 -enhancing contrast agents due to the abundant Fe 3+ on the surface of nanoparticles, while their T 2 effect decreases 13,14 .Usually, smaller Fe 3 O 4 nanoparticles can enhance the T 1 mode detection, and larger Fe 3 O 4 nanoparticles can improve the T 2 mode image due to their large innate high magnetic moment.The small size increases the surface-to-volume ratio of Fe ions, which improves their interaction with the surrounding water protons.The decrease in the magnetic core of superparamagnetic Fe 3 O 4 nanoparticles weakens the T 2 -weighted MRI response 15,16 .To enhance the T 2 effect, self-assembled ultrasmall Fe 3 O 4 nanoparticles were proposed to increase the magnetization.Therefore, some recent studies elaborately designed the surface chemistry of ultrasmall Fe 3 O 4 nanoparticles to make the particle size adjustable with some bioconditions, including specific pH 17,18 , hypoxia 19 , glutathione [20][21][22][23] and light 24 .However, some limitations still remain, such as complicated synthesis procedures, small production, and limited application conditions.Moreover, it is difficult to achieve homogeneity via the self-assembly of ultrasmall Fe 3 O 4 nanoparticles, which affects the signal reliability.More importantly, some designs enable T 1 to T 2 conversion but fail to simultaneously possess both T 1 and T 2 effects, which urges more devotion to developing ultrasmall Fe 3 O 4 nanoparticle-based T 1 /T 2 -weighted MRI contrast agents.Nanoparticles smaller than 5 nm can be facially cleared by the kidney, those 10-20 nm in size can be taken up rapidly, and the half-lifetime of the contrast agent in blood circulation was low 25,26 .The small size would make it easier for the nanoparticles to enter the tumor by permeation and retention effects, which would be beneficial for MRI examination.Therefore, the size control of the Fe 3 O 4 contrast agent should be considered an important factor.
Integrating high bioimaging capability and therapeutic efficacy simultaneously show promising perspective for developing new effective theranostic agent 27,28 .The ultrasmall size of Fe 3 O 4 nanoparticles not only endow the excellent MRI ability, but also enhance the exposed Fe ion content, which is essential for the CDT of tumor.The Fe 2+ on the surface of ultrasmall Fe 3 O 4 nanoparticles could serve as an effective catalyzer of high content H 2 O 2 in the tumor tissue to produce poison OH•, which could effectively induce apoptosis of cancer cell.Chemotherapy, one of the most used cancer treatments in clinic, suffers from no targeting and drug resistance.The collaborative effect of ultrasmall Fe 3 O 4 nanoparticles and chemotherapeutics could endow the MRI guided synergetic CDT and chemotherapy 29,30 .Moreover, integrated cancer cell targeting molecule could overcome the shortcoming of no targeting of therapeutic drugs.Therefore, the design and preparation of ultrasmall Fe 3 O 4 nanoparticle based nanomedicine for constructing tumor-targeted imaging and treatment nanoplatform in one system hold huge promising for resolving obstacle of tumor elimination 31 .
In the current work, we synthesized highly crystalline ultrasmall Fe 3 O 4 nanoparticle-based self-assembled nanoclusters with mass production for tumor-targeted dual-mode T 1 /T 2 -weighted MRI guided synergetic CDT and chemotherapy (Scheme 1).The ultrasmall Fe 3 O 4 nanoparticles were synthesized with a size of approximately 4 nm using a solvothermal method.After modification with 2,3-dimercaptosuccinic acid (DMSA), ultrasmall Fe 3 O 4 nanoparticles (Fe 3 O 4 -DMSA) can homogenously self-assemble into nanoclusters with several particles and maintain excellent dispersity and colloidal stability 32 .The Fe 3 O 4 -DMSA nanoclusters show excellent dual-mode T 1 /T 2 MRI property due to the favorable size distribution and could effectively induce apoptosis of cancer cell by CDT.Further conjugation with Arg-Gly-Asp (RGD) and doxorubicin (DOX), RGD ligand shows an improved binding affinity toward 4T1 cells through an overexpression of α v β 3 receptors 33 .Dox is an effective chemotherapy drugs for cancer cell.The treatment of self-assembled ultrasmall Fe 3 O 4 nanoclusters conjugated with RGD and DOX shows efficient T 1 /T 2 dual-mode MRI guided tumor-targeted synergetic CDT and chemotherapy.Therefore, this work provides a novel and convenient method to prepare integrated tumor-targeted MRI and nanodrugs treatment as well as proposes the concept of nanocluster-based functional MRI contrast agents and integrated treatment platform.

Instrument and characterization
The morphology of synthesized Fe 3 O 4 ultra-small nanoparticles and Fe 3 O 4 -DMSA nanoclusters was characterized by transmission electron microscopy (TEM; JEM-2100, JEOL, Tokyo, Japan), and the crystal structure of the Fe 3 O 4 nanoparticles was characterized by high-resolution transmission electron microscopy (HRTEM, JEM-2100, JEOL, Tokyo, Japan) and X-ray diffraction (XRD) patterns (D8 Advance, Bruker, Ettlingen, Germany).The element composition and atomic valence state were characterized by X-ray photoelectron spectroscopy (XPS).Fourier transform infrared (FTIR) spectra of the obtained nanomaterials were recorded by an IR spectrophotometer (Nicolet Nexus 670, Thermo Fisher Scientific, Inc., Waltham, MA).Ultraviolet-visible (UV-vis) light absorption spectra were obtained from a UV-vis spectrophotometer (UV-6100, Meipuda, Xi'an, China).The zeta potential and size distribution of the synthesized nanoparticles were characterized by a Malvern Zetasizer Nano Series.Element quantification was performed by an X-ray electron probe microanalyzer (EPMA).The

Mass production, modification and conjugation of ultra-small Fe 3 O 4 nanoparticles
The mass-production method of ultra-small Fe 3 O 4 nanoparticles was adapted from a previous report 34 .Briefly, all iron-oleate was obtained according to previous report 35 .Then, iron-oleate was added to 200 mL diphenyl ether and 50 mL oleyl alcohol.The mixture was heated to 200 °C and maintained for 30 min under N 2 atmosphere.The product was washed with ethanol and dried at 60 °C.The obtained brown powder was ultra-small Fe 3 O 4 nanoparticles.www.nature.com/scientificreports/Then, the nanoparticles were modified with DMSA molecules to synthesis Fe 3 O 4 -DMSA nanoclusters.Briefly, 20 mg of Fe 3 O 4 nanoparticles, 30 mg of DMSA, and 15 mg of Na 2 CO 3 were added to a mixture of tetrahydrofuran and water and ultrasonicated for 30 min.Afterwards, the mixture was collected by centrifugation and freezedried to obtain Fe 3 O 4 -DMSA nanoclusters.Fe 3 O 4 -DMSA nanoclusters can be mass-produced by amplifying this process tween times.
To conjugate the RGD ligand to synthesis Fe 3 O 4 -RGD nanoclusters, 4 mg aqueous Fe 3 O 4 -DMSA was added to the mixture of 1 mg of RGD, 2 mM EDC, and 4 mM S-NHS and reacted at 4 °C overnight.The product was dialyzed for 24 h.To synthesis Fe 3 O 4 -RGD-DOX, additional 0.2 mg of DOX was added to the same action media as RGD and the processing procedure was the same as the conjugation of RGD ligand.The aminofluorescein conjugation procedure was identical to the RGD.The bonding mechanism between Fe 3 O 4 and RGD as well as DOX are through the esterification between the COOH of Fe 3 O 4 -DMSA and NH 2 of DOX molecule and RGD ligand.

Cell lines and animal model
4T1 (mouse breast cancer) was obtained from the Institute of Biochemistry and Cell Biology (IBCB, Shanghai, China).The cells were cultured in RPMI-1640 culture medium with 10% fetal bovine serum (Gibco, USA) and 0.1% penicillin-streptomycin (Gibco, USA).Then, they were cultured in a humidified atmosphere at 37 °C in an incubator with 5% CO 2 .
All animal were purchased from the Jinan Peng Yue Laboratory Animal Co., Ltd, Jinan, China.The mice were fed with a standard laboratory diet and water.All mouse research were conducted in line with protocols approved by the Laboratory Animal Ethical and Welfare Committee of Shandong University Cheeloo College of Medicine, China (accreditation number: SYXK:20190005), and all animal experiments methods were performed according to these guidelines and regulations.To develop the 4T1 bearing tumor model, BALB/c male mice (6 to 8 week-old) were orthotopically injected with 1 × 10 6 4T1 cells in the right hind leg.

Tumor cell targeting assay of Fe 3 O 4 -RGD nanoclusters in vitro
To verify the RGD targeting effect, Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD were added to cultured 4T1 cells at 100 μg/ mL for 2 h.Then, the cancer cells were fixed with 2.5% glutaraldehyde and dehydrated in 30%, 50%, 70%, 80%, 90%, and 100% ethanol.The iron element content and distribution of the cells in each group were analyzed by EPMA.Additionally, Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD were conjugated with aminofluorescein and added to the cultured cells at a concentration of 100 μg/mL for 2 h.The fluorescence intensity of each group at 568 nm was observed by laser scanning confocal microscopy (LSCM, FV 300, Olympus, Japan).To verify that cellular uptake was receptor-specific, competition experiments were conducted using flow cytometry.The 4T1 cells were plated at 5 × 10 5 cells/ml in a 6-well plate and incubated with aminofluorescein-labeled Fe 3 O 4 -DMSA or Fe 3 O 4 -RGD solution.The cells were incubated for 12 h, washed twice with PBS, trypsinized, centrifuged and resuspended in 500 μl PBS before a flow cytometry analysis using a FACS Calibur flow cytometer (Becton Dickinson, NJ, USA).
Prussian blue staining kit were utilized to further detecte the RGD targeted effect for 4T1 cell uptake.Briefly, 4T1 cells were seeded in 24-well plates and incubated overnight.After washing with PBS twice, the cells were incubated with Fe 3 O 4 -DMSA (50 μg/mL, 100 μg/mL and 150 μg/mL) and Fe 3 O 4 -RGD (50 μg/mL, 100 μg/mL and 150 μg/mL).After 12 h cells were washed three times.Cells were fixed for 15 min in 4% paraformaldehyde, and then incubated for 25-30 min with 10% potassium ferrocyanide, rewashed twice with PBS, and counter stained with nuclear fast red for 10 min.Cells containing intracytoplasmic blue granules were defined to be Prussian blue staining positive.

Dual-mode T 1 /T 2 -weighted MR imaging ability of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters in vitro and vivo
For in vitro MRI, the 4T1 cells were washed with PBS and resuspended in 500 μL cell culture medium to verify the MRI effect at the cellular level by 3.0 T MRI scanner.T 1 -weighted imaging was performed with the following parameters: matrix size = 256 × 256, TR = 1200 ms, TE = 20 ms, slice thickness = 0.8 mm; T 2 -weighted imaging: matrix size = 256 × 256; TR = 2000 ms, TE = 62 ms; slice thickness = 0.8 mm.The T 2 -weighted signal change was calculated by using the following formula: SIi/SIc × 100% (i = 0.5, 1, 2, and 4 h), where SIc and SIi were the signal intensities of the 4T1 cells before and 0.5, 1, 2, and 4 h after incubation with Fe 3 O 4 -DMSA or Fe 3 O 4 -RGD, respectively.Additionally, the cultured cells in each group were stained with Prussian blue after 12 h of incubation with Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD according to the manufacturer instructions to confirm the RGD-enhanced targeting effect.

Biosafety evaluation of nanoparticles
To assess the hemolytic properties of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanomaterials, 2 mL of blood was dispersed to 4 mL of PBS (pH 7.4) and centrifuged at 1200 r/min for 10 min to isolate the red blood cells (RBCs).After washing with saline 3 times until the supernatant was colorless, the red blood cells were diluted with physiological saline to obtain 2% (v/v) red cell suspensions.Then, Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD at different final concentrations (25, 50, and 100 μg/mL) were co-incubated with red cell suspensions for 1 h at 37 °C.Simultaneously, equal volumes of distilled water and physiological saline were selected as positive and negative controls, respectively.After the incubation, the absorbance was measured by a microplate reader at 570 nm.The hemolytic degree was expressed by the hemolytic ratio using the following formula: hemolysis ratio To further detected the iron oxide distribution in vitro, 4T1 cells were incubated with PBS, Fe 3 O 4 -DMSA (100 μg/mL), Fe 3 O 4 -RGD (100 μg/mL), DOX (5 μg/mL) and Fe 3 O 4 -RGD-DOX (100 μg Fe 3 O 4 /mL, 5 μg DOX/ mL), after 12 h.Then Prussian blue staining were utilized to show positive 4T1 cells.The stain method were performed in agreement with previously described methods.Dichlorofluorescein diacetate (DCFH-DA) was used to verify the intracellular ROS.4T1 cells were treated with different concentrations of Fe 3 O 4 -DMSA (100 μg/ mL), Fe 3 O 4 -RGD (100 μg/mL), DOX (5 μg/mL) and Fe 3 O 4 -RGD-DOX (100 μg Fe 3 O 4 /mL, 5 μg DOX/mL) for 6 h.After that, the cells were washed with PBS and incubated with DCFH-DA solution (10 μM) for another 20 min.Afterwards, the cells were washed and then observed by CLSM.Mean intracellular fluorescence intensity was analyzed with the ImageJ software.

Dual-mode T 1 /T 2 -weighted MR imaging and synergistic effect of RGD targeting CDT and chemotherapy in vivo
To confirm the effective of MRI and combination therapy of CDT and chemotherapy.T 1 and T 2 weighted MR imaging were detected before and after intravenous injection of 100 µL normal saline, Fe 3 O 4 -DMSA (5 mg/ kg), Fe 3 O 4 -RGD (5 mg/kg), DOX (250 μg/kg) and Fe 3 O 4 -RGD-DOX (5 mg/kg Fe 3 O 4 /mL, 250 μg/kg Dox).The scanned parameters were performed in agreement with previously described methods.Coronal MRI data were collected pre-injection and post-injection.

Statistical analysis
All data were expressed as the average ± standard deviation.Statistical comparison between two groups was analyzed by the Student`s t-test.Analysis of variance (ANOVA) was used to compare the differences in different groups, and the data were defined with *p < 0.05, **p < 0.01 and ***p < 0.001.

Ethics statement
This study is reported in accordance with ARRIVE guidelines.

Synthesis and characterizations of ultra-small Fe 3 O 4 nanoparticles and Fe 3 O 4 nanoclusters
The mass production of nearly 2 g in one reaction of the synthesized Fe 3 O 4 nanoparticles is shown in Fig. S1, and the morphology was characterized by TEM (Figs. 1a, S2) and HRTEM (Figs. 1b, S3a).The obtained nanoparticles were of uniform shape, the size distribution was approximately 2-8 nm, and the main size was located at 4 nm (Fig. 1a, inset).The crystal plane spacings of 0.24 nm and 0.31 nm correspond to the (4 0 0) and (5 1 1) crystal planes, respectively, as shown in the HRTEM image.The well crystallinity of the individual ultra-small Fe 3 O 4 nanoparticles is confirmed, which benefits the increased saturation magnetization.During the modification process with DMSA molecules, the Fe 3 O 4 nanoparticles self-assemble into nanoclusters with 2-3 homogeneously embedded nanoparticles (Fig. 1c and the inset).Figure S4 confirms the stable synthesis and homogeneity of the Fe 3 O 4 -DMSA nanoclusters.The XRD pattern (Fig. 1d) of synthesized ultra-small Fe 3 O 4 nanoparticles confirms the standard Fe 3 O 4 phase (standard PDF card: 19-0629).The weak diffraction peak was assigned to the small size of the as-synthesized nanoparticles.Moreover, the synthesized nanoparticles were further analyzed by XPS (Fig. 1e,f).The spectrum of Fe 2p clearly shows the peaks of Fe 2+ and Fe 3+ , which confirms the composition of Fe 3 O 4 36 .The spectrum of O 1s contains the peaks assigned to Fe-O, C=O, and C-O, which indicates the coexistence of Fe 3 O 4 and oleic acid molecules.This is the key reason for the hydrophobic property of the as-synthesized Fe 3 O 4 nanoparticles and the requirement of DMSA surface modification.The saturation magnetization curve (Fig. 1g) measured by a vibrating sample magnetometer at room temperature confirms the superparamagnetic property of the obtained ultra-small Fe 3 O 4 nanoparticles with a large saturation magnetization (25 emu/g).The modification of Fe 3 O 4 with DMSA molecules was studied by FTIR spectroscopy (Fig. 1h).The broad peaks at 3297 cm −1 and 3252.5 cm −1 correspond to the stretching vibrations of O-H for oleyl alcohol and DMSA, respectively.The peaks at 1516.6 and 1402 cm −1 correspond to the symmetric and asymmetric stretches of carboxylate (COO-) in oleyl acid, while the peaks at 1567.4 and 1361 cm −1 correspond to the COO-in DMSA.All C-O stretching vibrations in oleyl alcohol and DMSA are at approximately 1045 cm −1 .The decreased C-H stretching vibrations at 2916.5 and 2848.1 cm −1 of oleyl alcohol indicate the substitution of DMSA at the surface of Fe 3 O 4 .Notably, the as-synthesized Fe 3 O 4 nanoparticles are spontaneously surface-modified by the organic solvent, which contributes to the good dispersion of Fe 3 O 4 nanoparticles in the organic solvent (Fig. S5).The surface hydrophilic treatment of nanoparticles is essential for the bio-application.XPS spectra of the Fe 3 O 4 -DMSA nanoclusters were also obtained, and the existence of S indicates surface modification by DMSA (Fig. S6).DOX molecule was confirmed to be conjugated on the surface of Fe 3 O 4 nanoclusters in the UV-vis absorbance result to constitute the nanomedicine (Fig. 1i).The size distribution of Fe 3 O 4 -DMSA is approximately 4.5-13.5 nm and the main size locates at 6 nm.(Fig. 1j).The conjugation of the RGD ligand and DOX molecule does not change the trend of the size distribution but increases the size by approximately 2 nm (Fig. 1k).Notably, the Fe 3 O 4 -DMSA nanoclusters show excellent colloidal stability.The nanocluster suspension in water remains clear and steady after being placed at 4 °C for 10 months and the size distribution (Fig. 1l) is identical to that of before.

Tumor cell targeting ability of Fe 3 O 4 -RGD nanoclusters
Nanomaterials usually possess passive targeting ability in tumor tissue due to the enhanced permeation and retention effect (EPR) and transport effect mediated by endothelial cells 37 .This ability is beneficial for the designed

T 1 /T 2 dual-mode MR imaging of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters both in vitro and vivo
The T 1 /T 2 dual-mode MRI of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters both in vitro and in vivo were furtherly studied.As shown in Fig. 3a, for T 1 -weighted imaging, the phantom images show enhanced lightness with increasing Fe 3 O 4 concentration.This result confirms that Fe 3 O 4 -DMSA nanoclusters possess the typical characteristics of T 1 contrast agents.For T 2 -weighted imaging, the phantom images tend to be darker when the Fe 3 O 4 concentration increases (Fig. 3b), which shows the typical characteristics of T 2 contrast agents.The T 1 -weighted and T 2 -weighted MRI signals of the gradient concentrations of Fe 3 O 4 nanoclusters indicate that the Fe 3 O 4 -DMSA nanoclusters is an excellent dual-mode T 1 and T 2 MRI contrast agent.The T 1 and T 2 relaxation times of the nanoclusters were simultaneously measured and are shown as T 1 and T 2 mappings according to the time horizon, respectively (Fig. 3c and d).The relaxivity values (r 1 and r 2 ) were obtained from the plots of inverse relaxation time (1/T 1 and 1/T 2 ) versus iron concentration (Fig. 3e and f).The calculated values of r 1 and r 2 are 0.296 mM −1 s −1 and 2.9 mM −1 s −1 , respectively, and the r 2 /r 1 value is 9.8.Generally, MRI contrast agents with a r 2 /r 1 ratio of approximately 5-10 are determined to be T 1 /T 2 dual-mode MRI contrast agents.Therefore, the synthesized Fe 3 O 4 -DMSA nanoclusters are excellent candidates of high-performance dual-mode contrast agents.The mechanism lies in that there still exist abundant Fe 3+ ions on the surface of self-assembled nanocluster, which endow the excellent T 1 mode MRI effect and the several nanoparticles self-assembled nanoclusters increase the equivalent volume of magnetic core, resulting in enhanced T 2 mode MRI effect 24,35 .
Based on the RGD targeting effect, the MRI effect of the designed nanoprobes were investigated on 4T1 cells before practical application in vivo.4T1 cells were cultured with Fe 3 O 4 -DMSA or Fe 3 O 4 -RGD nanoclusters at a concentration of 100 μg/mL for 0 h, 0.5 h, 1 h, 2 h, and 4 h.Then, the cells were digested, resuspended in PBS and investigated by the 3.0 T MR scanner.The T 1 -weighted images (Fig. 3g) show that the T 1 MRI signals became stronger with culture time for 4T1 cells cultured with either Fe 3 O 4 -DMSA or Fe 3 O 4 -RGD.Thus, more Fe 3 O 4 accumulates in 4T1 cells during the culture.The cells cultured with Fe 3 O 4 -RGD for various times showed a higher intensity of MRI response than those cultured with Fe 3 O 4 -DMSA at the same culture time because the targeting effect of RGD on 4T1 cells enhances the accumulation of Fe 3 O 4 nanoclusters.The statistical intensity data were analyzed as shown in Fig. 3h.The T 1 signal intensities were 104%, 115.7%, 129.3% and 157% in Fe 3 O 4 -DMSA and 109%, 130%, 149% and 210.6% in Fe 3 O 4 -RGD compared with the control group after culture with 4T1 cells for 0.5 h, 1 h, 2 h and 4 h, respectively.Since the T 2 mode MRI is a negative mode, the contrast agent reduces the signal.Similar to the T 1 -weighted images, the T 2 MRI signals (Fig. 3i) become weaker with increasing culture time for both 4T1 cells cultured with Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD.The cells cultured with Fe 3 O 4 -RGD for various  times show a weaker intensity of MRI response than those cultured with Fe 3 O 4 -DMSA at the same culture time.These results indicate a better T 2 mode MRI response with Fe 3 O 4 -RGD nanoclusters, which confirms the positive effect of the active targeting effect of RGD on 4T1 cells.The intensities are analyzed as shown in Fig. 3j.The T 2 signal intensities were 93.7%, 93.5%, 86.3% and 75.6% in Fe 3 O 4 -DMSA and 78.7%, 71.3%, 53.7% and 31.6% in Fe 3 O 4 -RGD compared with the control group after culture with 4T1 cells for 0.5 h, 1 h, 2 h and 4 h, respectively.The results show that the Fe 3 O 4 nanoclusters can actually serve as dual-mode T 1 /T 2 MRI contrast agents to largely strengthen the contrast at the cellular level.Moreover, the RGD ligand can remarkably increase the targeting effect of Fe 3 O 4 nanoclusters to form a faster and more high-intensity MRI response.
These Fe 3 O 4 -based nanoprobes were further investigated with 4T1 tumor-bearing mice in vivo.As shown in Fig. 3k and l, the T 1 and T 2 MRI images were acquired at different times after the intravenous injection of the Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoprobes.The effect of the contrast agent on T 1 -and T 2 -weighted images increases and decreases the MRI response, which are shown in red and blue, respectively.For the T 1 signals (Fig. 3k), the signal intensity at the tumor region in both Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD groups gradually increases during the first 4 h due to the rapid accumulation of Fe 3 O 4 nanoclusters but shows a slowed increasing tendency at the later time, which may be due to the metabolism in vivo and small accumulation amount of Fe 3 O 4 nanoclusters.At 4 h, the tumor tissue shows the best contrast to the surrounding tissues, which enables the diagnosis of the tumor tissue to be more accurate.Moreover, compared to the effect of Fe 3 O 4 -RGD with Fe 3 O 4 -DMSA, due to the targeting function of RGD to 4T1 cells, the tumor tissue of the Fe 3 O 4 -RGD group shows a higher contrast to the surrounding tissues than that of the Fe 3 O 4 -DMSA group at the same MRI examination time.Moreover, the enhanced contrast lasts longer for the Fe 3 O 4 -RGD group.Thus, the RGD targeting effect reduces metabolism in vivo.For T 2 -weighted imaging (Fig. 3l), the contrast enhancement of the tumor tissue was almost identical to the T 1 MRI result.The main difference may be that the T 2 contrast enhancement in Fe 3 O 4 -RGD is more obvious over time, which may be due to the constant aggregation effect of Fe 3 O 4 nanoclusters at the tumor site.These MRI results in vivo are mostly consistent with the in vitro results (Fig. 3g and i).To quantitatively analyze the contrast enhancement of T 1 and T 2 mode MRI, the signal intensity ratios SNRpost/SNRpre at different time points were analyzed and are shown in Fig. 3m and n, respectively.The T 1 signal intensities in the tumor sites were 113.8%, 118.1%, 138.1%, 121.9% and 107.3% in the Fe 3 O 4 -DMSA group and 120.8%, 136.4%, 149%, 141% and 95.6% in the Fe 3 O 4 -RGD group compared with the control group after 1 h, 2 h, 4 h, 8 h and 12 h of intravenous injection, respectively.The T 2 signal intensities in the tumor sites were 94.9%, 77.7%, 75.2%, 79% and 87.3% in the Fe 3 O 4 -DMSA group and 87.4%, 72.6%, 68.5%, 66.1% and 76.5% in the Fe 3 O 4 -RGD group compared with the control group after 1 h, 2 h, 4 h, 8 h and 12 h of intravenous injection, respectively.All of results confirm that Fe 3 O 4 -RGD can serve as an efficient activatable dual-mode MRI contrast agent for precise tumor diagnosis in vivo due to the ultra-small nanoparticle-based nanocluster structure and RGD targeting effect, which reveals its tremendous potential application for accurate tumor diagnosis in the clinic.
After the injection of the Fe 3 O 4 nanocluster-based nanoprobe, the in vivo iron distribution and biosafety were further investigated.Frozen sections of the tumor tissue were stained with Prussian blue to reveal the Fe distribution.As shown in Fig. S7, without Fe 3 O 4 injection, no Fe nanoparticles were present in the tumor tissue in the control.Some regions stained blue due to Fe accumulation after the intravenous injection of Fe 3 O 4 -DMSA.In contrast, large areas were stained blue with the intravenous injection of Fe 3 O 4 -RGD.This result further confirms the targeting effect of RGD and the induced improved accumulation property of Fe 3 O 4 -RGD.In addition, the Fe 3 O 4 -DMSA nanoclusters are dispersed in the tumor tissue with no mass aggregation, but most of the Fe 3 O 4 -RGD nanoclusters aggregate near the blood vessels, which indicates that the RGD ligand can efficiently target the α v β 3 receptors of endothelial cells in the tumor site and tumor cells.Furthermore, ultrathin sections of tumor tissue were examined by TEM (Fig. S8).Fe 3 O 4 -based nanoclusters were detected at the tumor sites in both groups.To understand the metabolism of the Fe 3 O 4 nanoclusters, the amount of Fe in the main organs and tumors in vivo was analyzed by ICP-MS measurements (Fig. S9).The Fe ion concentrations in the heart, spleen, lung and kidney hardly change with or without the intravenous injection of Fe 3 O 4 nanoclusters.The Fe concentration significantly increases from ~ 100 to ~ 400 μg/g in the liver, which indicates that the liver is the main uptake organ and key metabolic organ for Fe 3 O 4 nanoclusters.Notably, the Fe concentration in tumors increases with the application of Fe 3 O 4 nanoclusters.Moreover, the Fe 3 O 4 -RGD group was almost 4 times the control and 3 times the Fe 3 O 4 -DMSA group.This result is attributed to the great targeting effect of RGD on 4T1 cells.Good hemocompatibility is essential for nanomaterials for biomedical applications in vivo.The hemocompatibility of the Fe 3 O 4 nanocluster-based nanoprobe was evaluated through a hemolytic assay (Fig. S10).Compared with the effects of water (positive control) and physiological saline (negative control), no distinct hemolysis could be observed with either Fe 3 O 4 -DMSA or Fe 3 O 4 -RGD over the studied concentration range.The quantitatively analyzed hemolysis percentages of these nanoclusters were less than 2%, even when the concentration increased to 100 μg/mL These results suggest that the designed nanoprobe exhibits excellent hemocompatibility.

Fe 3 O 4 nanoclusters based nanomedicine for T 1 /T 2 dual-mode MR imaging and synergistic CDT and chemotherapy
Before the Fe 3 O 4 nanoclusters being used as the MRI contrast agent and chemotherapy drug carries of tumor in vivo, 4T1 cells were firstly cultured with Fe 3 O 4 -DMSA nanoclusters at different concentrations to evaluate the CDT effect.The living/dead staining results (Fig. S11) show that there were fewer living cells with increasing Fe 3 O 4 concentration and culture time.The CCK-8 results (Fig. S12) show the same trend of growth inhibition and toxicity effects on the tumor cells.Moreover, the cytotoxicity of Fe 3 O 4 -RGD shows distinct differences (in the concentration of 50 μg/mL, p = 0.017; 100 μg/mL, p = 0.003 and 150 μg/mL, p < 0.001) compared with www.nature.com/scientificreports/nanoclusters (Fig. S13).When cultured with 50 μg/mL Fe 3 O 4 , the ROS level was much higher than that of the control.In addition, it is furtherly enhanced with 150 μg/mL of Fe www.nature.com/scientificreports/ the tumor targeting ability of RGD ligand and the DOX conjugation possesses no effect on the targeting effect.Furtherly, the digested tumor cells were imaged with both T 1 and T 2 weighted MR (Fig. 6b) and the signal statistics were shown in Fig. 6c and d  tumor growth via the combinational effect from the chemotherapy and CDT.In addition, as shown in the Fig. 7c, the adopted treatment for mice would not influence the body weight, indicting the suitable dose and strategy of tumor therapy.In addition, the Prussian blue staining (Fig. 7d) and statistical result (Fig. 7h) show that the group of Fe 3 O 4 -RGD and Fe 3 O 4 -RGD-DOX have more Fe accumulation in tumor tissue.Hematoxylin/eosin (H&E) (Fig. 7e) and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining with statistical result (Fig. 7f and i) show significant tumor cell death and the formation of many cavities, indicating that the Fe 3 O 4 -RGD-DOX can effectively induce tumor tissue damage.Meanwhile, the immunhistochemical studies (Fig. 7g and j) manifest that Fe 3 O 4 -RGD-DOX led to the most caspase3 expression, indicating that Fe 3 O 4 -RGD-DOX could efficiently induce tumor cell apoptosis, which well supported the in vitro anticancer result.All in all, these results indicated that the RGD meditated tumor targeted synergistic treatment effect of chemotherapy and CDT induced by ultrasmall Fe 3 O 4 nanoclusters and DOX hold great tumor-growth inhibition effect.

Biosafety evaluation after synergetic theranostics
After the in vivo antitumor study, the H&E staining of main organs including heart, liver, spleen, lung and kidney were used to evaluated biocompatibility and histological variations of Fe 3 O 4 nanocluster-based nanomedicine was evaluated through blood test.Moreover, the main blood indexes of mice treated with Fe 3 O 4 -RGD-DOX as well as other treatment at different days were further analyzed, showing no obvious change in all groups (Fig. 8b-e).These preliminary results show that the designed nanomedicines possess excellent biocompatibility in vivo.

Conclusion
In summary, an effective method of mass produce ultra-small Fe 3 O 4 nanoparticles was proposed.The further surface modification with DMSA endows the hydrophilic property and high colloidal stability in suspension as well as the self-assembled property.The obtained Fe 3 O 4 nanoclusters show an excellent T 1 /T 2 dual-mode MRI capacity and excellent potential for use as contrast agents due to the appropriate size effect.In addition, the ultrasmall Fe ion concentration was quantified by inductively coupled plasma-mass spectrometry (ICP-MS).The saturation magnetization of Fe 3 O 4 nanoparticles was measured by a vibrating sample magnetometer (MicroMagTM Model 2900 AGM system).The relaxation rate of MRI images of the Fe 3 O 4 nanoprobe was tested by a 3.0-T clinical MRI scanner (GE Signa HDx 3.0 T MRI, USA) and a 16-channel brain coil.

Fe 3 O 4 -
= (O.D of sample − O. D of negative control)/(O.D of positive control − O. D of negative control) × 100%.RGD-DOX nanomedicine for T 1 /T 2dual-mode MR imaging and synergistic CDT and chemotherapy in vitroThe RGD targeted CDT effect of Fe 3 O 4 nanoclusters was furtherly studied.4T1 cells were treated by the coincubation of Fe 3 O 4 -DMSA (2.5, 5, 10, 20, 50, 100 and 150 μg/mL) and Fe 3 O 4 -RGD (2.5, 5, 10, 20, 50, 100 and 150 μg/mL) for 24 h.Then, the cell viability was determined using the CCK-8 assay (n = 5).The 4T1 cells were seeded in 24 cell culture plates, and a designed concentration of Fe 3 O 4 -DMSA was added to the culture medium for a specific time.Live/dead staining was performed to evaluate the cytotoxicity of the nanoclusters.Additionally, ROS staining by DCFH-DA was performed to briefly demonstrate the reason for toxicity after the 2 h co-culture of Fe 3 O 4 at 50 μg/mL, 150 μg/mL and cancer cells.To further verify the synergistic chemotherapy and CDT induced by Fe 3 O 4 -RGD-DOX nanomedicine, 4T1 cells were exposed to different concentrations of Fe 3 O 4 -DMSA (100 μg/mL), Fe 3 O 4 -RGD (100 μg/mL), DOX (5 μg/mL) and Fe 3 O 4 -RGD-DOX (100 μg Fe 3 O 4 /mL, 5 μg DOX/mL) and further measured by Living/dead staining and CCK-8 test.Untreated cells in the medium were used as a control.Corresponding groups without cells were used as blanks.The cell viability was calculated by assuming 100% viability in the control cells.The proliferation ability was analyzed through an EdU Proliferation Kit.Briefly, 4T1 cells grown in 96-well plates were subjected to different concentrations of Fe 3 O 4 -DMSA (100 μg/mL), Fe 3 O 4 -RGD (100 μg/mL), DOX (5 μg/ mL) and Fe 3 O 4 -RGD-DOX (100 μg Fe 3 O 4 /mL, 5 μg DOX/mL) for 24 h.Then EdU was incorporated into the treated cells and detected through a catalyzed reaction with a fluorescently labeled azide and were observed by fluorescence microscopy.

Fe 3 O 4 -
DMSA nanocluster.However, due to the low half-lifetime of the contrast agent in blood circulation, passive targeting is limited.The active targeting ability of Fe 3 O 4 -DMSA nanoclusters is endowed by conjugation with the tumor targeting RGD ligand.It is expected to improve the accumulation of Fe 3 O 4 nanoclusters in the tumor region and realize a favorable contrast effect.As shown in Fig. 2a, the iron element content was analyzed by EPMA after the 4T1 cells were cultured with Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD for 2 h.More iron was detected in the Fe 3 O 4 -RGD group than in the Fe 3 O 4 -DMSA group, which indicates the better accumulation property of Fe 3 O 4 -RGD nanoclusters.Furthermore, the aminofluorescein molecule was conjugated with the Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters to locate the nanoparticles by fluorescence.As shown in Fig. 2b, the 4T1 cells were cocultured with fluorescence-labeled Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters and assayed by flow cytometry.The results convey that more cells were labeled with fluorescence in the Fe 3 O 4 -RGD group than in the Fe 3 O 4 -DMSA group, which shows that the RGD ligand enhances the cell uptake ability.Then, fluorescencelabeled Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters were cultured with 4T1 cells at a concentration of 100 μg/ mL for 2 and 4 h.As shown in Fig. 2c, after the culture with Fe 3 O 4 -RGD, the green fluorescence intensities were higher than those cultured with Fe 3 O 4 -RGD for 2 and 4 h.This result confirms the enhanced targeting effect of the RGD ligand.Prussian blue staining was utilized to further study the existence of iron in 4T1 cells after culturing with Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters at various concentrations for 12 h.As shown Fig. 2d, the staining results are consistent with the discussion above.The tumor cells cultured with Fe 3 O 4 -RGD can accumulate more Fe 3 O 4 nanoclusters than that cultured with Fe 3 O 4 -DMSA.These results confirm that the RGD ligand can enable an active targeting effect toward tumor cells.

Figure 2 .
Figure 2. Tumor cell targeting ability of Fe 3 O 4 -RGD.(a) Iron element content distribution on the surface of 4T1 cells after being cultured with Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters (color scale bar shows the Fe element content).(b) Flow cytometry assay of 4T1 cells after incubation with fluorescence labeled Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters.(c) Fluorescence labeling of 4T1 cells with Fe 3 O 4 -aminofluorescein and Fe 3 O 4 -RGD-aminofluorescein for 2 h.(d) Prussian blue staining of 4T1 cells after being cultured with different concentrations of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters for 12 h.

Figure 3 .
Figure 3. T 1 /T 2 dual-mode MRI of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters both in vitro and vivo.(a) T 1 -weighted MR images, (c) T 1 mapping and (e) T 1 relaxation time of Fe 3 O 4 -DMSA nanoclusters at various iron concentrations.(b) T 2weighted MR images, (d) T 2 mapping and (f) T 2 relaxation time of Fe 3 O 4 -DMSA nanoclusters at various iron concentrations.(g) T 1 MRI images of 4T1 cells cultured with Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters and (h) the corresponding signal intensity statistics.(i) T 2 MRI images of 4T1 cells cultured with Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters and (j) the corresponding signal intensity statistics.(k) T 1 MRI images of tumors in vivo at different times after the injection of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters.(l) T 2 MRI images of tumors in vivo at different times after the injection of Fe 3 O 4 -DMSA and Fe 3 O 4 -RGD nanoclusters.(m) T 1 -and (n) T 2 -weighted MRI signal intensity statistics at different times.

Figure 6 .
Figure 6.Tumor targeted T 1 and T 2 MRI images of tumor cells and tumors as well as the signals statistic.(a) Prussian blue staining, (b) The T 1 and T 2 MRI image and (c), (d) the corresponding MRI signals statistic of digested 4T1 cells after incubation with Fe 3 O 4 nanoclusters based nanomedicine.(e) The T 1 and T 2 MRI image and (f,g) the corresponding MRI signals statistic of tumor tissue before and after intravenous injection of Fe 3 O 4 nanoclusters based nanomedicines.

Figure 7 .
Figure 7.In vivo antitumor and biosafety evaluation of tumor targeted synergistic CDT and chemotherapy.(a) Real tumor image after the collaborative treatment.(b) Tumor volume of tumor bearing mice during the collaborative treatment.(c) Body weight of tumor bearing mice during the collaborative treatment.(d) Prussian blue staining, (e) HE staining, (f) TUNEL staining, (g) Caspase 3 staining of tumor tissue sections after the collaborative treatment.(h) The statistics of Fe ion expression obtained from Prussian blue staining (i) the statistics of positive expression of TUNEL.(j) The statistics of positive expression of Caspase 3.

Figure 8 .
Figure 8. Biosafety evaluation of collaborative treatment of Fe 3 O 4 nanoclusters based nanomedicine.(a) Images of hematoxylin and eosin (H&E)-stained histological tissue sections of major organs.(b-e) Main blood indexes of mice during the collaborative treatment.
3 O 4 nanoclusters possess notable CDT effect caused by the increased ROS level in tumor cells.The collaborative work of chemotherapy drugs DOX and Fe 3 O 4 nanoclusters base CDT treatment endow highly effective antitumor activity for tumor treatment.With the assistance of RGD targeting ligand, the integrated Fe 3 O 4 -RGD-DOX nanoplatform possesses tumor targeted T 1 /T 2 dual-mode MRI guided synergistic CDT and chemotherapy, which provides a novel practical alternative for integrated diagnosis and treatment of tumors and exhibits promising potential for clinical translation.
The MR imaging of mice were obtained at pre-injection and at 1 h, 2 h, 4 h, 8 h and 12 h post-injection.After MR scanning, slices covering the entire tumor region were prepared.TEM, ICP-MS and Prussian blue stained were utilized to verify the MRI results.